Chemistry & Biochemistry Theses and Dissertations
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Item Kinetic Characterization and Domain Analysis of the helicase RecD2 from Deinococcus radiodurans(2010) Shadrick, William Robert; Julin, Douglas A; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The gram positive bacterium D. radiodurans is known for its extreme resistance to radiation and an extraordinary ability to reconstitute its genome after sustaining large numbers of double strand breaks (DSB's). Genome analysis does not immediately reveal a biochemical basis for this incredible DNA repair ability. In E. coli, DSB's are mainly repaired through the RecBCD pathway via homologous recombination. The D. radiodurans genome contains no known homologues of RecB or RecC, but sequence analysis has identified a homologue of RecD, termed RecD2. The function of RecD2 in D. radiodurans is unknown, as RecD elsewhere has only been found as a component of the RecBCD complex. Our research has focused on biochemical characterization of RecD2. Previous work in our lab established that RecD2 is a DNA helicase with limited processivity and a preference for forked substrates. We have studied the unwinding mechanism of the enzyme, as measured by rates of DNA unwinding and behavior on various substrates. Reactions conducted under single turnover conditions have allowed us to determine the processivity and the step size of RecD2. RecD2 pre-bound to dsDNA substrate is capable of unwinding 12 bp, but not 20 bp, when excess ssDNA is added to prevent rebinding of enzyme to substrate. Unwinding of the 12 bp substrate under single turnover conditions could be modeled using a two step mechanism, with kunw = 5.5 s-1 and dissociation from partially unwound substrate koff = 1.9 s-1. Results derived from these rate constants indicate an unwinding rate of 15-20 bp/ sec, with relatively low processivity (P = 0.74). Glutaraldehyde cross-linking showed formation of multimers of RecD2 in the absence of DNA, but this was not detectable by size exclusion chromatography. We were able to separate the N-terminal region from the helicase core of RecD2 using limited proteolysis. It was not possible to characterize the C-terminal helicase domain due to its low solubility upon overexpression in E. coli.Item BIOCHEMICAL AND BIOLOGICAL CHARACTERIZATION OF THREE DNA REPAIR ENZYMES IN DEINOCOCCUS RADIODURANS(2009) Cao, Zheng; Julin, Douglas A; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The Gram positive bacterium Deinococcus radiodurans is able to withstand acute doses of gamma rays that can cause hundreds of double-strand breaks per genome. In proposed double-stand break repair pathways, however, some important enzymes, such as helicases and nucleases in the initiation step, have not been clearly identified yet. Interestingly, the common bacterial helicase/nuclease complex RecBCD or AddAB, which functions to produce a 3' ssDNA tail in double-strand break repair initiation step in other bacteria, is not found in D. radiodurans. As part of efforts to identify helicases involved in double-strand break repair, the D. radiodurans HelIV (encoded by locus DR1572, the helD gene) was characterized with both in vivo and in vitro methods. The helD gene is predicted to encode a helicase superfamily I protein. The helD mutant is moderately sensitive to methyl methanesulfonate and hydrogen peroxide but it is not sensitive to gamma rays, UV and mitomycin C. In biochemical assays, the full length HelIV exhibited DNA unwinding activity with a 5'-3' polarity whereas the truncated HelIV without N-terminal region had no detectable helicase activity. RecJ is the exonuclease in the RecF pathway, which is suggested to function at the initiation step in DSB repair in the absence of RecBCD. In the in vivo study, the D. radiodurans recJ gene (encoded by locus DR1126) cannot be completely removed from the chromosome, indicating the essential role of RecJ in cell growth. The heterozygous mutant displayed growth defect and higher sensitivity to gamma rays, hydrogen peroxide and UV compared to wild type D. radiodurans, suggesting an important role in DNA repair. The RecJ expressed in E. coli system was insoluble but can be purified via denaturation-refolding, and the refolded RecJ showed 5'-3' exonuclease activity. D. radiodurans has no RecB and RecC proteins, but it has a homologue of the RecD protein. We tested whether the D. radiodurans RecD protein could form a complex or make transient interactions with other proteins to perform more complicated functions. The RecD conjugated protein affinity column was used to attempt to identify cellular binding partners.Item PURIFICATION AND CHARACTERIZATION OF THE RECD PROTEIN-HOMOLOGUE FROM DEINOCOCCUS RADIODURANS(2004-12-06) Wang, Jianlei; Julin, Douglas A; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In many gram-negative bacteria, RecBCD enzyme is found to be responsible for double strand DNA break repair through homologous recombination. The AddAB enzyme, a RecBCD analog, is found in some gram-positive bacteria and functions in a similar way as RecBCD. A few bacteria appear to lack both RecBCD and AddAB enzymes entirely. One such organism is the bacterium Deinococcus radiodurans. This remarkable organism is able to survive in the presence of very high levels of radiation or DNA-damaging chemicals, levels that would overwhelm the DNA repair capacity of most other organisms. Interestingly, the D. radiodurans genome does have an open reading frame that would encode a protein that is homologous to the E. coli RecD protein. The amino acid sequence of this D. radiodurans RecD-like protein suggests that it is a helicase and therefore could function in some aspect of DNA repair, as does its E. coli homologue. However, the RecD protein of D. radiodurans must serve a different and novel function compared to the E. coli RecD protein. The D. radiodurans RecD protein can be expressed at high levels in E. coli and is readily purified by chromatography on a nickel column followed by single-stranded DNA-cellulose. The purified protein exhibits DNA-dependent ATPase and DNA helicase activities. The helicase activity requires at least a 10 nucleotide single strand overhang at the 5'-end of the double strand DNA substrate to start unwinding. The helicase assay shows that D. radiodurans RecD-like protein unwinds dsDNA substrates catalytically, but with low processivity, even with the help of single strand binding proteins (SSB) from either E. coli or D. radiodurans. These results show that D. radiodurans RecD-like protein is a DNA helicase that moves with 5'-3' polarity on single-stranded DNA. The E. coli RecD protein was shown recently to unwind dsDNA with the same 5'-3' polarity. The low processivity of the D. radiodurans RecD-like protein suggests that it may function in a complex with other proteins. The identity of these proteins is not known. We have also generated insertion mutations that are likely to disrupt all of the recD gene copies in the D. radiodurans genome after multiple generations growing in media with antibiotics. The in vivo effects of the insertion mutation, such as the growth curve and the sensitivity to UV radiation and DNA damaging chemicals, were studied.